NO168086B - PROCEDURE FOR PREPARING A STABILIZED Aqueous INSULIN SOLUTION - Google Patents

Description

The present invention relates to a method for the production of hitherto unknown physically stabilized insulin solutions.

Insulin that is dissolved in a liquid medium, e.g. water,

can be stored for several years at room temperature, and the preparations are stable within this period. If, however, one heats an insulin solution to approx. 80°C, the insulin denatures within a few minutes, which is referred to here as heat denaturation or heat polymerisation. If one shakes an insulin solution for a few days at a lower temperature where no, or i

essentially none, heat denaturation takes place, e.g. at 41°C, a different kind of polymerization will take place, and this polymerization is referred to here as non-covalent border f ] c-te polymerization.

At insulin manufacturers, insulin retailers and patients, insulin preparations are usually stored at approx. 5°C, and that

apparently no interfacial polymerization occurs in such preparations, even though the preparations are inevitably shaken during transport from time to time.

During the last few years, an increasing effort has been made to develop portable or implantable systems for the continuous infusion of insulin. The mechanical part of a device

for continuous delivery of insulin in principle includes such components as an insulin reservoir, a pump system and a suitable catheter for delivering insulin to the patient. If the insulin solution is supplied by a pump, this can also act as an insulin reservoir.

When insulin in commercially available solutions is placed in such systems, it has unfortunately been shown that the insulin has a tendency to undergo interfacial polymerization at body temperature and thus clog both mechanical parts and delivery catheters. This characteristic feature of insulin solutions has proven to be a major obstacle to further development and clinical application of apparatus for continuous infusion.

It is clear that insulin solutions in any type of continuous delivery apparatus are subject to motions that produce interfacial polymerization. The general imperfection of known insulin preparations in this context is amply documented in the literature, see e.g. Diabetologia 19 (1980),

To solve this problem, it has been proposed to use acidic insulin solutions containing glutamic acid or aspartic acid, cf. Diabetes 30 (1981), 83. However, insulin is chemically unstable in acid, even at temperatures below body temperature. It has also been proposed to use insulin preparations containing non-ionic surfactants, cf. West German patent application P 2,952,119. However, non-ionic surfactants can be considered undesirable in pharmaceuticals for parenteral use.

These problems are overcome by the present invention

which relates to a method for the production of hitherto unknown, dissolved insulin preparations, in which the insulin is significantly less inclined to undergo non-covalent interfacial polymerization under the conditions that exist in continuous insulin delivery apparatus than is the case with conventional insulin preparations.

It has now surprisingly been shown that insulin solutions are stabilized against interfacial polymerization when there is at least one phospholipid present in the solution.

According to the invention, a method is provided for the production of a stabilized, aqueous insulin solution which optionally contains a further component selected from among zinc salts, preservatives, agents which make the solution isotonic, and buffers, and where the insulin is essentially outside liposomes. The method is characterized by insulin being mixed with water and the other components of the solution and with a stabiliser, the insulin possibly being added as the last component and the solution being subjected to ultrasound treatment in advance or an insulin solution is mixed with a stabilizer, preferably with an aqueous stabilizer solution, which is optionally subjected to ultrasound treatment,

in that a phospholipid with the general formula I is used as a stabilizer

where R' and R" are the same or different and each represents hydrogen, alkylcarbonyl, alkenylcarbonyl, alkadienylcarbonyl, alkatrienylcarbonyl or alkatetraenylcarbonyl, with at least one of R' and R" having a meaning other than hydrogen, and R"'

denotes a hydrophilic group.

Preferred examples of R"' are 2-(trimethylammonio)-

ethyl, 2-aminoethyl, 2-carboxy-2-aminoethyl, 2,3-dihydroxypropyl or 2,3,4,5,6-pentahydroxycyclohexyl. The above-mentioned alkylcarbonyl, alkenylcarbonyl, alkadienylcarbonyl, alkatrienylcarbonyl and alkatetraenylcarbonyl groups can contain from 8 to 22 carbon atoms, and in one embodiment of this invention they contain from 12 to 22 carbon atoms.

A preferred subgroup of compounds of formula I are compounds where - R' and R" each denote alkylcarbonyl.

A further preferred subgroup of compounds of formula I are compounds where R"' denotes 2-(trimethylammonio)-ethyl, which compounds are known as lecithins. Further preferred are compounds of formula I where R<1> and R" each denotes alkylcarbonyl with from 12 to 16 carbon atoms, especially from 8 to 16 carbon atoms, and R"' denotes 2-(trimethylamino)ethyl. The most preferred subgroup of compounds of formula I are compounds in which R<1> and R" each denotes octanoyl. Compounds of formula I where R' and R" each denote octanoyl are preferred, because they do not appear to form liposomes^ and further because at lower concentrations, e.g. below about 160 µq/ml, they do not appear to form micelles .

The amount of a phospholipid of formula I necessary for stabilization of eh insulin solution is preferably in the range from 10 to 200 µg/ml, more preferably from 10 to

100 µg/ml, preferably from 25 to 75 µg/ml, and

most preferably from 30 to 50 ug/ml insulin solution.

The concentration of dissolved insulin in the solutions produced by the method according to the present invention is in the range from 5 to 1000 international units (IU) per ml or even higher.

Some of the stabilized insulin solutions prepared according to the following examples may contain liposomes. However, it is unlikely that such liposomes contain insulin, as they are formed before the addition of insulin. If there are liposomes present in the insulin solutions produced by the method according to the second invention, it is preferred that the insulin is essentially outside such liposomes. The phrase "substantially" means that preferably more than 901, and preferably more than. 99% of the insulin is outside any liposomes present.

Known liposomes containing insulin, see e.g. European patent application 32,622, differs from this invention both by their purpose and because the presence of insulin inside a possibly present liposome vesicle in the insulin solutions produced by the method according to the present invention is purely accidental.

While the insulin solutions produced by the method according to the present invention are intended for parenteral administration, oral administration is the primary reason for encapsulating insulin in liposomes. One purpose of liposomes containing insulin is to protect the insulin against unwanted chemical attack, e.g. chemical breakdown of the insulin in the stomach, if the insulin is administered orally. The articles relating to liposomes containing insulin do not deal in any way with physical stabilization of insulin solutions against interfacial polymerization. In known liposomes containing insulin, the weight ratio between the phospholipid and insulin is e.g. in the range from 1:0.01 to 1:0.001, while the weight ratio for the insulin solutions produced by the method according to the present invention is in the range from 1:5 to 1:10,000,

preferably from 1:10 to 1:1000.

West German official letter 2. 652,636 relates to a method for stabilizing sensitive proteins by adding protective compounds with an amphophilic structure. In contrast to the present invention, the stabilization according to the above-mentioned publication is achieved by encapsulating the sensitive protein to avoid contact with water. Furthermore, according to the above-mentioned publication's terminology, insulin is not to be considered a sensitive protein.

One purpose of this invention is to keep the insulin in contact with water, i.e. in solution, the insulin being excluded from contact with other interfaces.

The insulin solutions produced by the method according to the present invention preferably contain bovine, porcine or human insulin.

According to a preferred embodiment, the insulin solutions produced by the method according to the present invention contain zinc. However, the amount of zinc must be chosen so that no precipitation occurs. A good stability against interfacial polymerization is achieved in insulin solutions where the ratio between the molar concentration of zinc ions available for the insulin, and the molar concentration of insulin calculated as hexameric insulin, is in the range from

1.5 to 4.6, preferably from 3 to 4.5,

most preferably from 3.6 to 4.3. Preferred zinc salts are soluble zinc salts such as zinc acetate or zinc chloride.

If the insulin solutions produced by the method according to the present invention contain compounds that form complexes with insulin, such as an amino acid, e.g. glycine or histidine, or a hydroxycarboxylic acid, e.g. citric acid,

is only part of the total zinc content available to the insulin.

An example of the preparation of insulin solutions according to the invention comprises dissolving insulin, e.g. crystalline zinc insulin, e.g. highly purified insulin such as "monocomponent" insulin, cf. British patent 1,285,023, in water in the presence of acid, e.g. hydrochloric acid. An aqueous solution of a preservative, e.g. phenol, an alkylphenol such as cresol, or p-hydroxybenzoic acid methyl ester, and the solution also contains, if desired, an agent that makes the solution isotonic, such as sodium chloride or glycerol. The preservative solution may also contain a

buffer such as sodium chloride, sodium citrate, sodium acetate or TRIS (tris(hydroxymethyl)amihometane). The resulting preservative solution is then optionally added to the acidic insulin solution, followed by the addition of a base, e.g. a sodium hydroxide solution, to adjust the pH to neutral. In the context of this invention, the term neutral pH indicates a pH value in the range from approx. 6.5 to approx. 8. The phospholipid of formula I can be added to the insulin solution as a solution or colloidal solution prepared by dissolving or suspending the phospholipid of formula I in water and orr. necessary to subject any suspension to ultrasound treatment before mixing with the insulin solution. The phospholipid cj' solution ke.r. if desired contain a buffer and a preservative. After mixing in the phospholipid, the insulin preparation's pH value can be set to neutral. The resulting insulin solution is finally filled up to the calculated volume by adding

settling of water, then sterilized by filtration and then aseptically transferred to sterile vials which are then closed.

The present invention therefore relates to a method for producing insulin solutions, which method is characterized by mixing insulin with a compound of formula I and optionally with a zinc salt, a preservative, an agent that makes the solution isotonic and a buffer in the presence of water.

Some of the compounds of formula I are known, and the remaining compounds of formula I can be prepared analogously to the preparation of known compounds.

Further details for carrying out the present invention are given in the following examples, which should not, however, be interpreted as limiting the scope of the invention in any way. The insulin starting material used in the examples contains from 20 to 35 ug zinc per mg nitrogen. The determination of the stability factor appears from the following.

To determine the insulin solutions' stability against interfacial polymerization, the solutions are subjected to a stability test under forced conditions in the following way: 12.5 ml vial containing 10 ml test sample. each provided with a rubber stopper and placed vertically in a shaker apparatus (sold by Heto, Denmark) which is totally immersed in a water bath, which is maintained at 41°C 1 0.1°C. The vials are subjected to horizontal rocking movements with a frequency and amplitude of 100 rpm and 50 mm respectively.

The opalescence of the test samples is determined at regular time intervals on a "Fischer DRT 1000 nephelometer" (sold by Fischer, Canada). Interfacial polymerization is assumed to have taken place when the turbidity exceeds 10 NTU (nephelometric turbidity units).

The stability factor is calculated as the ratio between the time dc-jt that elapses before interface denaturation occurs in the test sample,

and the corresponding time for a control sample that has been prepared

• in the same way as the test sample, with the proviso that no compound of formula I has been added.

Example 1

500 mg of semi-synthetic human insulin is dissolved in 10 ml

0.045N hydrochloric acid, and 359 mg of p-hydroxybenzoic acid methyl ester dissolved in 300 ml of distilled water is added. Furthermore, 476 mg of sodium acetate trihydrate, 2.46 g of sodium chloride are added

and 4.73 ml of a 0.2N sodium hydroxide solution dissolved in

15 ml of distilled water. 9 mg dimyristoyl,L-alpha-phosphatidylcholine is suspended in 10 ml of a solution of 70 mg sodium chloride,

- 13.6 mg sodium acetate and 10 mg p-hydroxybenzoic acid methyl ester

in distilled water. Nitrogen is bubbled through the slurry,

which is treated with ultrasound in an ultrasound bath for 2 hours. The resulting colloidal solution is added with stirring to the insulin solution. The pH value is adjusted to 7.45 with 0.2N hydrochloric acid or a 0.2N sodium hydroxide solution, and distilled water is added to a volume of 350 ml. The resulting insulin solution stability factor is above 125.

Example 2

9.65 g porcine insulin is dissolved in 400 ml 0.02N hydrochloric acid,

5.0 g of crystalline phenol and 40 g of anhydrous glycerol are added, and further distilled water is added to a volume of 2200 ml. The pH value is adjusted to 7.45 with a Q.2N sodium hydroxide solution. 125 mg of distearoyl, L-alpha-phosphatidylcholine is dissolved by gentle heating in 2 ml of ethanol (96%) and injected with vigorous stirring via a needle into 100 ml of distilled water at a temperature of 70°C.

The resulting cloudy solution is treated for 15 min. sonicated with a high-energy ultrasound probe, the resulting colloidal solution is stirred into the insulin solution, and distilled water is added to a volume of

2500 ml. If necessary, the pH value is set to 7.45. The stability factor is over 30.

Example 3-8

Insulin solutions are prepared in an analogous manner as described in example 1, with the proviso that the phospholipids used are leitins, where the hydrophobic parts, i.e. R' and R" are identical and are as indicated in table I. The results also appear in table I.

Example 9

An insulin solution is prepared in an analogous manner to that described in example 1, with the proviso that the phospholipid used is egg lecithin and the insulin used is pig insulin. The stability factor is 96.

Example 10-14

Insulin solutions are prepared in an analogous manner as described in example 1 with the proviso that. porcine insulin is added in an amount which gives the final concentrations indicated in Table II. The results are also shown in Table II.

Oak sample 15

1.50 g of porcine insulin is dissolved in 6.5 ml of 0.2N hydrochloric acid, and water is added to a volume of 50 ml. 1.0 g of p-hydroxybenzoic acid methyl ester and 1.78 g of sodium phosphate are dissolved in 900 ml of distilled water, and the insulin solution is added. The pH value is adjusted to 7.45 with a 0.2N sodium hydroxide solution.

A colloidal dimyristoyl, L-alpha-phosphatidylcholine solution, which is prepared in an analogous manner as described in Example 2, is added together with distilled water to a volume of 1000 ml for

to achieve a final phospholipid concentration of 50 pg/ml.

The stability factor is over 30.

Example 16

An insulin solution is prepared in an analogous manner as described in example 15 - with the proviso that the final solution contains 20 IU insulin per ml. The stability factor is over 17.

Example or 17

An insulin solution is prepared in an analogous manner as described in example 15 with the proviso that the final insulin concentration is 500 IU/ml and the final content of dimyristoyl, L-alpha-phosphatidylcholine is 50 ug/ml - The stability factor is over 30.

E xample 18-20

Insulin solutions are prepared in an analogous manner as described in example 2, with the proviso that dimyristoyl, L-alpha-phosphatidylcholine is used in such an amount that the final concentration indicated in Table III is achieved. The results also appear in table III..

Example 21

Dioctanoyl, L-alpha-phosphatidylcholine is dissolved in distilled water and added in an amount sufficient to obtain

a final concentration of 30 µg/ml, to an insulin solution prepared analogously to the insulin solution in example 1. The stability factor is above 63.

. Example 22

3.65 g of semi-synthetic human insulin is dissolved in 100 ml of 0.02N hydrochloric acid and 2.0 g of crystalline fendl. 16 g of anhydrous glycerol and 0.3 ml of zinc chloride solution containing 4% zinc are added, and further distilled water is added to a volume of 900 ml. The pH value is adjusted to 7.45 with a 0.2N sodium hydroxide solution. Dissolve 50 mg of dioctanoyl, L-alpha-phosphatidylcholine in distilled water and add to the solution. Distilled water is added to a volume of 1000 ml. The stability factor is 65.

Claims (7)

1. Method for the production of a stabilized aqueous insulin solution which possibly contains an additional component selected from zinc salts, preservatives, agents that make the solution isotonic, and buffers, and where the insulin is essentially outside liposomes, characterized by insulin being mixed with water and the other components of the solution and with a stabilizer, with the insulin possibly being added as the last ingredient and the solution is subjected to ultrasound treatment in advance or an insulin solution is mixed with a stabilizer, preferably with an aqueous stabilizer solution, which is optionally subjected to ultrasound treatment, the stabilizer being used a phospholipid of the general formula I where R' and R" are the same or different and each represents hydrogen, alkylcarbonyl, alkenylcarbonyl, alkadienylcarbonyl, alkatrienylcarbonyl or alkatetraenylcarbonyl, wherein at least one of R' and R" has a meaning other than hydrogen, and R<1>" denotes a hydrophilic group. 2. Method according to claim 1, characterized in that a compound of formula I is used where R<1>" is 2-(trimethylammonio)ethyl, 2-aminoethyl, 2-carboxy-2-aminoethyl, 2,3-dihydroxypropyl or 2,3,4,5,6-pentahydroxycyclohexyl. 3. Method according to any one of the preceding claims, characterized in that a compound of formula I is used where R"' denotes 2-(trimethylammonio)-ethyl. 4. Method according to claim 3, characterized in that a compound of formula I is used where R' and R" each denote alkylcarbonyl containing from 8 to 16 carbon atoms. 5. Method according to claim 4, characterized in that a compound of formula I is used where R' and R" each denote alkylcarbonyl containing from 12 to 16 carbon atoms. 6. Method according to any one of claims 1-4, characterized in that a compound of formula I is used where R' and R" each denote octanoyl. 7. Method according to any one of the preceding claims, characterized in that a phospholipid of formula I is used together with at least one zinc salt, the ratio between the molar concentration of zinc ions available for the insulin and the molar concentration of insulin calculated as hexameric insulin, is in the range of 1.5 to 4.6.